NMDA receptors with restricted mobility of the ligand binding domain

描述（由申请人提供）：NMDA受体是膜结合的神经递质受体，在脑生理学中起关键作用。它们是治疗阿尔茨海默病、中风、智力迟钝、成瘾、慢性疼痛、精神分裂症和癫痫的主要药物靶点。NMDA受体是谷氨酸激活的兴奋性离子通道。当神经递质谷氨酸与细胞外配体结合域（lbd）结合时，它们的激活反应就开始了，并随着膜嵌入的阳离子选择性孔的打开而达到高潮。构成NMDA受体激活反应的分子事件仍然未知，尽管它们在控制受体功能方面起着基本作用。单受体活性的动力学研究已经证实，在结合激动剂后，NMDA受体以可测量的速率在几种闭孔和开孔构象之间循环。对分离的LBD的结构研究表明，配体结合在两个活动叶之间的缝隙深处，并提出了配体诱导的LBD闭合是NMDA受体激活途径的必要步骤之一的假设。本应用程序提出的实验将直接和具体地通过检查NMDA受体的活性与受限的lbd的流动性来测试这一假设。对半胱氨酸残基将被引入到lbd叶的尖端，作为一种手段，将它们锁定在相对于彼此的规定距离上。工程受体将通过动力学分析在存在和不存在氧化剂的情况下的宏观和单通道行为，以及用不同长度的交联试剂处理后的行为来检验。这些结果将提供与NMDA受体激活相关的分子运动的基本信息，并将为药物作用于该受体的可能机制提供有价值的见解。这些知识将形成必要的概念基础，以合理控制NMDA受体活动，作为中风、成瘾和精神疾病的治疗策略。公共卫生相关性：该项目将提供有关NMDA受体激活机制的基本信息。这些信息目前是缺乏的，而对于急性和慢性神经变性（中风、阿尔茨海默病）、成瘾、慢性疼痛、认知障碍和精神疾病的新治疗方法的合理设计是需要的。

DESCRIPTION (provided by applicant): NMDA receptors are membrane-bound neurotransmitter receptors with critical roles in brain physiology. They are principal drug targets for Alzheimer's disease and stroke, mental retardation, and also addiction, chronic pain, schizophrenia and epilepsy. NMDA receptors are glutamate-activated, excitatory ion-channels. Their activation reaction is initiated when the neurotransmitter glutamate binds to extracellular, ligand-binding domains (LBDs) and culminates with the opening of a membrane-embedded cation-selective pore. The molecular events that make up the activation reaction of NMDA receptors remain unknown, despite their fundamental role in controlling receptor function. Kinetic studies of single-receptor activity have established that after binding agonists, NMDA receptors cycle with measurable rates between several closed-pore and open-pore conformations. Structural studies of the isolated LBDs have shown that ligands bind deep inside a crevice between two mobile lobes and have put forth the hypothesis that ligand-induced closure of the LBD is one of the required steps along the NMDA receptor activation pathway. This application proposes experiments that will directly and specifically test this hypothesis by examining the activity of NMDA receptors with restricted mobility of the LBDs. Pairs of cysteine residues will be introduced at the tips of the LBDs lobes as a means to lock these at defined distances with respect to one another. The engineered receptors will be examined by kinetic analyses of their macroscopic and single-channel behaviors in the presence and absence of oxidizing agents, and following treatment with crosslinking reagents of various lengths. The results will provide basic information regarding the identity of the molecular motions associated with NMDA receptor activation and will afford valuable insight regarding possible mechanisms of drug action at this receptor. This knowledge will form the conceptual foundation necessary to rationally control NMDA receptor activities as a therapeutic strategy for stroke, addiction, and mental illness. PUBLIC HEALTH RELEVANCE: This project will provide fundamental information regarding the mechanism by which NMDA receptors become active. This information is currently lacking and is needed for the rational design of novel therapeutic approaches for acute and chronic neurodegeneration (stroke, Alzheimer's disease), addiction, chronic pain, cognitive disorders and mental illness.